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Abstract

This work was focused on the quantitative analysis of time-resolved in vitro measurements of ligand-induced ERBB signalling in breast cancer cell lines, as well as the development of experimental methods suitable for the large-scale analysis of signalling networks. First, an automated protocol for the highly reproducible stimulation of cell lines with growth factors was developed. In parallel, protein microarray technologies were advanced to the quantification of phosphoproteins and resulted in two different assay formats: microspot immunoassays and reverse phase protein arrays. In collaboration with the bioinformatics group, data analysis tools were developed for both platforms. Experiments in ERBB2 overexpressing cell lines, HCC1954 and SKBR3, demonstrated that both ERBB2 targeting monoclonal antibodies, trastuzumab and pertuzumab, did not efficiently prevent ligand-induced signalling in vitro. Moreover, the combination of both antibody therapeutics did not result in improved efficacy. However, combining a single therapeutic antibody with the EGFR inhibiting small molecule erlotinib significantly downregulated ligand-induced signalling. Furthermore, treatment of proliferating cells with the combination of trastuzumab and erlotinib resulted in a dephosphorylation of the ribosomal protein S6 and the cell cycle regulator protein RB resulting in cell cycle arrest. Thus, the combination of erlotinib with trastuzumab could be postulated as potential therapy for the treatment of ERBB2-positive breast cancer patients. A comparative analysis of ERBB signalling in four cell lines, MCF7, BT474, HCC1954, and SKBR3, revealed that the phosphorylation of the ribosomal protein S6 is a strong predictor to analyse the activation status of signalling networks since the S6 protein integrates signals from the MAPK as well as the PI3K pathway, the two major pathways downstream of ERBB receptors. Due to differential ERBB receptor expression or additional oncogenic mutations, therapeutics affected ERK1/2 and AKT signalling to different extents in the four cell lines whereas the S6 phosphorylation reflected reliably the cellular response on exogenous perturbations.